This year we have continued to identify novel mAbs in several formats as Fabs, scFvs and eAds against cancer-related proteins mostly for pediatric patients. These mAbs were tested for their activity against cancer cells in vitro and in vivo and used for development of novel approaches for multispecific targeting. Two of the mAbs in CAR format, against CD22 and mesothelin, entered clinical trials. We have also characterized drugability of some mAbs including their propensity for aggregation. The major accomplishments are summarized below. 1) T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor beta (FR) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Our mAb m909 was used to develop the first CARs specific for human FR which was evaluated in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FR as well as endogenous FR(+) AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FR(+) THP1 AML in vivo. In addition, our collaborators demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FR expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), the FR expression and recognition of HSCs by CAR T cells was also evaluated. m909 CAR T cells were not toxic against healthy human CD34(+) HSCs in vitro. These results indicate that FR is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA. 2) The expression of FR was also evaluated in other cancer cells. Over-expression of folate receptor alpha on cancer cells has been frequently exploited for delivery of folate-targeted imaging and therapeutic agents to tumors. Because limited information exists on expression of the beta isoform of the folate receptor in most human cancers (FR), the immunohistochemical staining pattern of FR in 992 tumor sections from 20 different human cancer types was evaluate using our anti-human FR mAb m909. FR expression was shown to be more pronounced in cells within the stroma, primarily macrophages and macrophage-like cells than cancer cells in every cancer type studied. Moreover, FR expression in both cancer and stromal cells was found to be statistically more prominent in females than males. A significant positive correlation was also observed between FR expression on stromal cells and both the stage of the cancer and the presence of lymph node metastases. Based on these data we conclude that FR may constitute a good target for specific delivery of therapeutic agents to activated macrophages and that accumulation of FR positive macrophages in the stroma could serve as a useful indicator of a tumor's metastatic potential. 3) The insulin-like growth factors (IGFs), IGF-1 and IGF-2, have been implicated in the growth, survival and metastasis of a broad range of malignancies including pediatric tumors. They bind to the IGF receptor type 1 (IGF-1R) and the insulin receptor (IR) which are overexpressed in many types of solid malignancies. Activation of the IR by IGF-2 results in increased survival of tumor cells. We have previously identified a novel human monoclonal antibody, m708.5, which binds with high (pM) affinity to both human IGF-1 and IGF-2, and potently inhibits phosphorylation of the IGF-1R and the IR in tumor cells. m708.5 exhibited strong antitumor activity as a single agent against most cell lines derived from neuroblastoma, Ewing family of tumor, rhabdomyosarcoma and osteosarcoma. When tested in neuroblastoma cell lines, it showed strong synergy with temsirolimus and synergy with chemotherapeutic agents in vitro. In xenograft models, the combination of m708.5 and temsirolimus significantly inhibited neuroblastoma growth and prolonged mouse survival. Taken together, these results support the clinical development of m708.5 for pediatric solid tumors with potential for synergy with chemotherapy and mTOR inhibitors. 4) We have previously proposed an approach for fast and irreversible removal of IGF2 from the circulation by using monoclonal antibodies (mAbs) that bind to two or more non-overlapping epitopes on the same molecule. We provided initial evidence for the formation of oligomeric antibody-ligand complexes that can bind to cells expressing Fc gamma receptors (FcgamaRs) with high avidity using an antibody domain with relatively low affinity as one of the anti-IGF2 mAbs. Recently, we identified a mAb, m708.5, in a scFv format which binds to both IGF2 and IGF1 with very high (pM) affinity. Interestingly, and rather surprisingly, this mAb did not compete with our other high affinity mAb, m610.27, for binding to IGF2. Therefore, we generated a new bispecific mAb, m67, by combining m708.5 and m610.27. As expected m67 potently inhibited binding of IGF2 to cells expressing the IGF1R and its phosphorylation, and resulted in formation of multimolecular complexes when incubated with IGF2 and bound with high avidity to cells expressing FcgamaRII; the complexes were internalized in a macrophage-like cell line. However, although m67 exhibited a reasonably long half-life (6.4 days) in cynomolgus macaques and high stability in serum, its administration to three animals did not result in any measurable decrease in the IGF2 concentration likely due to the complexity of the IGF2 interactions in the blood and the relatively low (2mg/kg) dose of the mAb leading to a relatively low maximal blood concentration of 120 nM. In spite of the lack of effect on the IGF2 concentration in this particular experimental setup, m67 exhibited good drugability properties and could be highly effective in other animal models and in humans. Studies with animal models of cancer are ongoing to evaluate the potential of m67 as a new candidate mAb-based therapeutic. 5) Glypican-3 is a cell surface glycoprotein that associates with Wnt in liver cancer. Our collaborators developed two antibodies targeting glypican-3, HN3 and YP7; HN3 was derived from our antibody domain library. HN3 recognizes a functional epitope and inhibits Wnt signalling, whereas YP7 recognizes a C-terminal epitope but does not inhibit Wnt signalling. Both are fused to a fragment of Pseudomonas exotoxin A (PE38) to create immunotoxins. Interestingly, the immunotoxin based on HN3 (HN3-PE38) has superior antitumor activity as compared with YP7 (YP7-PE38) both in vitro and in vivo. Intravenous administration of HN3-PE38 alone, or in combination with chemotherapy, induces regression of Hep3B and HepG2 liver tumour xenografts in mice. This study establishes glypican-3 as a promising candidate for immunotoxin-based liver cancer therapy. Our results demonstrate immunotoxin-induced tumour regression via dual mechanisms: inactivation of cancer signalling via the antibody and inhibition of protein synthesis via the toxin. 6) Currently, we participate in the work of the Pediatric Oncology Dream team where we identified and performed initial characterization of several mAbs against targets of importance for childhood cancers. The results will be reported in the next year report after additional characterization of those antibodies including in animal models.